Histidinol dehydrogenase (L-histidinol: NAD+ oxidoreductase, HDH; EC 1.1.1.23) is one of three biosynthetic enzymes encoded by the tri-functional histidine biosynthesis (his) gene in high fungi, which catalyzes the last two steps of the histidine biosynthesis. A single histidinol dehydrogenase (Hdh2) gene was identified in wheat-biotype Phaeosphaeria nodorum (PN-w) and Phaeosphaeria sp. from Polish rye (P-rye). The Hdh2 gene was not present in barley-biotype P. nodorum (PN-b), P. avenaria f. sp. avenaria (Paa), 3 groups of P. avenaria f. sp. triticea (Pat1, Pat2 and Pat3) and Phaeosphaeria sp. from dallis grass (Paspalum dilatatum Poir.). Since the Hdh2 gene in PN-w Sn37-1 isolate was not expressed in the cultures grown in rich and minimal media, it was a pseudo-gene. Most of the amino acids important for substrate binding and Zn2+ ligand formation for histidinol dehydrogenase enzymatic activity were well conserved in the Hdh2 protein. In phylogenetic analysis based on the deduced peptide sequences, the Hdh1 peptides in the HIS proteins of the fungi were grouped with the bacteria which had cysteine (C) at both C-116 and C-153 positions in histidinol dehydrogenases. The Hdh2 proteins from 3 Aspergillus and 2 Phaeosphaeria species were grouped with the bacteria, which had mostly leucine (L) at C-116 and all with valine (V) at C-153 positions in histidinol dehydrogenases. Evolution of the Hdh2 genes in Phaeosphaeria and Aspergillus species were discussed.